Søren Bregenholt

The Role of Up-Regulated Serine Proteases and Matrix Metalloproteinases in the Pathogenesis of a Murine Model of Colitis

Proteinases are important at several phases of physiological and pathological inflammation, mediating cellular infiltration, cytokine activation, tissue damage, remodeling, and repair. However, little is known of their role in the pathogenesis of inflammatory bowel disease. The aim of this study was to assess the role of tissue proteases in a mouse model of colitis. Proteolytic activity was analyzed, using gel and in situ zymography, in colonic tissues from severe combined immunodeficient mice with colitis induced by transfer of CD4(+) T lymphocytes. Serine proteinase levels increased in colitic tissue, with major species of 23 kd, 30 kd, and 45 kd. Co-migration and inhibition studies indicated that the 23-kd proteinase was pancreatic trypsin and that the 30-kd species was neutrophil elastase. Matrix metalloproteinase (MMP)-9 expression, and MMP-2 and MMP-9 activation, was elevated in colitic tissues. Proteinase levels followed a decreasing concentration gradient from proximal to distal colon. Proteolysis was localized to infiltrating leukocytes in diseased severe combined immunodeficient mice. Transmural inflammation was associated with serine proteinase and MMP activity in overlying epithelium and with marked subepithelial proteolytic activity. The results demonstrate a clear elevation in the levels and activation of proteases in colitis, potentially contributing to disease progression through loss of epithelial barrier function.

Invariant Vα14+ NKT Cells Participate in the Early Response to Enteric Listeria monocytogenes Infection

Invariant Vα14+ NKT cells are a specialized CD1-reactive T cell subset implicated in innate and adaptive immunity. We assessed whether Vα14+ NKT cells participated in the immune response against enteric Listeria monocytogenes infection in vivo. Using CD1d tetramers loaded with the synthetic lipid α-galactosylceramide (CD1d/αGC), we found that splenic and hepatic Vα14+ NKT cells in C57BL/6 mice were early producers of IFN-γ (but not IL-4) after L. monocytogenes infection. Adoptive transfer of Vα14+ NKT cells derived from TCRα° Vα14-Jα18 transgenic (TCRα°Vα14Tg) mice into alymphoid Rag°γc° mice demonstrated that Vα14+ NKT cells were capable of providing early protection against enteric L. monocytogenes infection with systemic production of IFN-γ and reduction of the bacterial burden in the liver and spleen. Rechallenge experiments demonstrated that previously immunized wild-type and Jα18° mice, but not TCRα° or TCRα°Vα14Tg mice, were able to mount adaptive responses to L. monocytogenes. These data demonstrate that Vα14+ NKT cells are able to participate in the early response against enteric L. monocytogenes through amplification of IFN-γ production, but are not essential for, nor capable of, mediating memory responses required to sterilize the host.

Signal transduction by the major histocompatibility complex class I molecule

Ligation of cell surface major histocompatibility class I (MHC‐I) proteins by antibodies, or by their native counter receptor, the CD8 molecule, mediates transduction of signals into the cells. MHC‐I‐mediated signaling can lead to both increased and decreased activity of the MHC‐I‐expressing cell depending on the fine specificity of the anti‐MHC‐I antibodies, the context of CD8 ligation, the nature and cell cycle state of the MHC‐I‐expressing cell and the presence or absence of additional cellular or humoral stimulation. This paper reviews the biochemical, physiological and cellular events immediately after and at later intervals following MHC‐I ligation. It is hypothesized that MHC‐I expression, both ontogenically and in evolution, is driven by a cell‐mediated selection pressure advantageous to the MHC‐I‐expressing cell. Accordingly, in addition to their role in T‐cell selection and functioning, MHC‐I molecules might be of importance for the maintenance of cellular homeostasis not only within the immune system, but also in the interplay between the immune system and other organ systems.

In vitro activated CD4+ T cells from interferon-gamma (IFN-γ)-deficient mice induce intestinal inflammation in immunodeficient hosts

To investigate the role of IFN‐γ in the immunopathogenesis of inflammatory bowel disease (IBD), severe combined immunodeficient (SCID) mice were transplanted with in vitro activated CD4+ T cells from either wild‐type (WT) or IFN‐γ‐deficient (IFN‐γKO) BALB/c mice. In vitro, the two types of T cells displayed comparable proliferation rates and production of tumour necrosis factor‐alpha (TNF‐α), IL‐2, IL‐4 and IL‐10 after concanavalin A (Con A) stimulation. When transplanted into SCID mice, WT CD4+ blasts induced a lethal IBD, whereas IFN‐γKO blasts induced a less severe intestinal inflammation with moderate weight loss. Intracellular cytokine staining of lamina propria lymphocytes (LPL) revealed comparable fractions of CD4+ T cells positive for TNF‐α, IL‐2 and IL‐10 in the two groups of transplanted SCID mice, whereas a two‐to‐three‐fold increase in the fraction of IL‐4‐positive cells was found in IFN‐γKO‐transplanted SCID mice. Flow cytometric analyses showed strong up‐regulation of MHC class II expression of colonic epithelial cells of WT‐CD4+ T cell‐transplanted compared with IFN‐γKO‐transplanted SCID mice. A significantly higher fraction of CD4+ LPL were found to enter the cell cycle, i.e. to incorporate bromo‐dexoy‐uridine, and to undergo apoptosis in vivo in WT‐transplanted compared with IFN‐γKO‐transplanted SCID mice. These data point towards an important role for IFN‐γ in the development of IBD in SCID mice. The inflammation might be initiated and subsequently enhanced by the ability of IFN‐γ to induce denovo MHC class II expression in the colonic epithelium, a change which could lead to increased antigen processing and production of local proinflammatory cytokines, CD4+ T cell turnover and thereby to exaggeration of disease.

Proliferation and apoptosis of lamina propria CD4 + T cells from scid mice with inflammatory bowel disease

Scid mice transplanted with low numbers of syngeneic CD4+ T cells, develop a chronic and lethal inflammatory bowel disease (IBD) within 4 – 6 months. We have used in vivo 5‐bromo‐2‐deoxy‐uridine (BrdU) labeling to assess the proliferation of lamina propria‐derived CD4+ T cells in diseased scid mice. The hourly rate of renewal of colonic lamina propria CD4+ T cells in diseased mice was 7 % compared with 1.5 % in normal BALB/c control mice. Transplantation of scid mice with in vitro activated CD4+ T cells accelerated the disease onset and development in a cell dose‐dependent fashion when compared with non‐activated CD4+ T cells. In pulse‐chase experiments it was shown that BrdU‐labeled cells disappeared rapidly from the lamina propria of diseased mice. DNA analysis revealed that this was due to the presence of nearly four times as many apoptotic CD4+ T cells in diseased than in control mice. Further analyses showed that the apoptotic lamina propria CD4+ T cells were derived from cells having entered the cell cycle within the previous 8 h. These data clearly demonstrate that vigorous CD4+ T cell proliferation and death are involved throughout the course of IBD.

Cells and Cytokines in the Pathogenesis of Inflammatory Bowel Disease: New Insights from Mouse T Cell Transfer Models

Recently, a number of experimental models of human inflammatory bowel disease (IBD) of immunological basis have been developed. These have proven useful tools in the study of IBD, allowing a more detailed dissection of the pathogenesis of the disease. Studies from these models have revealed new, important knowledge about environmental factors, cell subset, cytokines and effector molecules in the pathogenesis of IBD. This review focuses on recent advances in the understanding of the development of IBD obtained from adoptive CD4+ T cell transfer models of the disease.

Accumulation of immunoglobulin-containing cells in the gut mucosa and presence of faecal immunoglobulin in severe combined immunodeficient (scid) mice with T cell-induced inflammatory bowel disease (IBD)

Scid mice transplanted either with a gut wall graft or with low numbers of purified CD4+ T cells from immunocompetent syngeneic donor mice show clinical signs of IBD 3–4 months post‐transplantation. The disease is mediated by mucosa‐infiltrating CD4+ TCRαβ+ T cells. The pathology of 52 individual colon segments obtained from 20 gut wall‐ or CD4+ T cell‐transplanted diseased scid mice was evaluated by histology and the numbers of infiltrating immunoglobulin‐containing cells were determined. In particular, cells positive for IgM, IgA and non‐inflammatory immunoglobulin isotypes such as IgG1 and IgG2b were found to accumulate in colon segments displaying the most severe histopathology, including inflammatory cellular infiltration, epithelial hyperplasia and ulcerative lesions. Compared with colon segments of normal C.B‐17 mice, the lesional scid colon shows increased levels of cells positive for the IgG classes. Faecal extracts of the CD4+ T cell‐transplanted scid mice revealed the presence of all six murine immunoglobulin isotypes. Disease progression was accompanied by an increased level of excreted IgM and IgG3 and decreased levels of IgA. It is concluded that locally secreted immunoglobulins may play an immunomodulating role in the pathological changes observed in the present model of T cell‐induced inflammatory bowel disease.

The majority of lamina propria CD4+ T-cells from scid mice with colitis undergo Fas-mediated apoptosis in vivo

We have previously shown that adoptively transferred CD4(+) T-cells mediate an chronic colitis in severe combined immune deficient (scid) mice. Colitis is accompanied by activation and apoptosis of Fas ligand and TNF-alpha expressing CD4(+) T-cells in the diseased colonic lamina propria (Eur. J. Immunol. 28:3655 (1998)). Here we investigate the apoptosis-inducing mechanism in these lamina propria infiltrating CD4(+) T-cells. We observe that freshly isolated lamina propria CD4(+) T-cells can kill Fas transfected P815 mastocytoma cells in a TCR/CD3 redirected chromium-release assay, but do not express TNF-alpha mediated cytotoxicity. Pre-incubation of the isolated lamina propria CD4(+) T-cells with an anti-FasL antiserum partially blocked killing of the Fas transfected target cells, indicating a role for the Fas-FasL system in the killing process. Treatment of scid mice with colitis with anti-FasL antiserum for 12 h blocked the apoptotic process in lamina propria CD4(+) T-cells by more than 65% compared to mice treated with control antiserum. Together, these results point towards the Fas-FasL and not the TNF-alpha-TNF-alpha receptor system as the primary apoptosis-inducing mechanism of lamina propria CD4(+) T-cells in this model of murine chronic colitis, and suggest an important role for the Fas-FasL system in the maintenance of homeostasis of locally proliferating T-cells.

No evidence for altered cellular immune functions in personnel deployed in the Persian Gulf during and after the Gulf War--The Danish Gulf War study.

Veterans who have participitated in the Gulf War suffer from a number of symptoms, collectively referred to as the Gulf War Syndrome. It has been hypothesized that a change in the systemic cytokine balance or other changes in immunological parameters could be responsible for some of the symptoms. We analyzed the peripheral blood natural killer (NK) cell activity of 686 Gulf War personnel who had been present in the Persian Gulf area during and immediately after the Gulf War as well as 231 gender and age‐matched controls. The test material included individual samples of frozen peripheral blood mononuclear cells kept at −139°C for a period of 50 to 380 days prior to NK cell analysis of freshly thrawed cells. Significant differences in NK‐cell activity were not observed by direct comparison of the levels of natural cytotoxic activity in the two groups. However, NK‐cell cytotoxicity as such decreased due to cryopreservation. Surprisingly, the NK cells obtained from control donors were significantly (p<0.0001) more sensitive to freezing conditions than cells from the Gulf War personnel, leaving the marginal comparison between the two groups untrustworthy, in particular because of the marked difference between the −139°C storage times used for the two groups. Freshly thawed samples of peripheral blood T lymphocytes (CD2+ cells) from 109 randomly selected Gulf War personnel and 68 gender‐ and age‐matched controls were stimulated for 3 days with phytohemagglutinin followed by 4 h activation by phorbol ester and ionomycin, and were stained for intracellular content of interleukin‐2, ‐5, ‐10 and interferon‐γ. As with natural cytotoxicity, the length of cell storage at −139 °C influenced the production of cytokines. No significant differences in the cytokine production between the two groups were observed when the influence of the storage period was taken into consideration. Together, these data suggest that no overall long‐term effects on NK‐cell function and T‐cell cytokine production are present in the Danish Gulf War personnel. Moreover, cryopreservation is a major potential source of bias when studying the physiology of thawed NK and T cells.

Tumour necrosis factor-alpha (TNF-α) transcription and translation in the CD4+ T cell-transplanted scid mouse model of colitis

The adoptive transfer of activated CD4+α/β T cell blasts from the spleens of immunocompetent C.B‐17+/+ or BALB/cdm2 mice into C.B‐17scid/scid (scid) mice induces a colitis in the scid recipient within 8 weeks, which progresses to severe disease within 16 weeks. T cells isolated from recipient colon show a Th1 cytokine phenotype. We have examined the relationship between the phenotype of the cellular infiltrate and the transcription and translation of the proinflammatory cytokine TNF‐α. The techniques of double indirect immunohistology and in situ hybridization using digoxigenin‐labelled riboprobes were used. The prominent myeloid cell infiltrate in diseased tissues comprised F4/80+, Mac‐l+ macrophages, neutrophils, dendritic cells and activated macrophages. TNF‐α transcription and translation were associated with activated macrophages in the lamina propria. Activated macrophages transcribing and translating TNF‐α were clustered in areas of tissue destruction. Crypt epithelium of inflamed tissues transcribed TNF‐α at a very early stage of the disease process, but translation of TNF‐α protein could only be found in advanced epithelial dysplasia. This indicates differential post‐transcriptional control of TNF‐α in activated macrophages and the epithelium.